Phosphorylation is a ubiquitous post-translational protein modification that plays a critical role in a variety of cellular functions. In fact, various drugs targeting the kinases that phosphorylate protein substrates have been developed to treat diseases from cancers to Alzheimer's disease. As a result, the monitoring of kinases and their phosphoprotein substrates in cells represents an important goal towards characterizing healthy and disease states and development of new drugs. Unfortunately, available methods to monitor cellular phosphorylation are challenging. New methods for monitoring and charactering kinases and their modified substrates are necessary to allow a full understanding of cellular signaling and disease formation. The long-term goal of this project is a rigorous characterization of kinase activity and kinase-catalyzed modifications. The scientific foundation of the project is our recent revelation that kinases promiscuously accept ?-phosphate modified ATP analogs as cosubstrates. Based on kinase ATP cosubstrate promiscuity, we are pursuing two significant aims. First, we hypothesize that kinases catalyze protein modifications in addition to phosphorylation. Our hypothesis is based on the fact that several ?-phosphate modified ATP analogues are naturally occurring and associated with cell stress conditions, although their mechanisms of action are unknown. Consistent with our hypothesis, we have recently discovered a novel post-translational protein modification, termed nucleotide phosphorylation. With their key role in cell signaling, these studies are critical to illuminate the overall role of kinases in cell biology. Our second significant aim centers on experiments to label kinase substrates with various functional probes to allow detection, sequence identification and characterization of the kinase-catalyzed modifications. By using the developed chemical approaches to monitor and characterize the role of kinases in healthy and diseased tissues, the significant outcome of the overall project is a greater understanding of signaling in disease formation. Because kinase enzymes are targeted in drug development, these studies will lead in the creation of more effective drugs influencing protein phosphorylation. PUBLIC HEALTH RELEVANCE: Kinase enzymes are involved in various diseases and are the target of multiple pharmaceutical drugs. Kinases phosphorylate protein substrates, making a characterization of phosphoproteins an important aspect of health-related research. The application outlines new approaches to characterizing kinase substrates and kinase-catalyzed modifications to aid in the understanding of disease and development of new treatments.